Epithelial Ion and Fluid Transport Flashcards
Why does water movement across epithelia matter
- body temperature regulation
- mucus movement (pathogen clearance from lung)
- renal fluid balance
- digestion and nutrient absorption
- reproduction
- diarrhoea (pathogen clearance from gut)
Daily oral fluid input
2L
Daily saliva fluid input
1.5L
Daily gastric juice fluid input
2.5L
Daily bile fluid input
0.5L
Daily pancreatic fluid input
1.5L
Daily intestinal fluid input
1L
Daily total fluid input
9L
How much fluid is lost in faeces
0.1L
How much fluid is recovered by the small intestine
7L
How much fluid is recovered by the large intestine
1.9L
Action of cholera
- inhibits fluid reabsorption in gut
- epithelial function was measured by checking how much fluid was in bucket
The transepithelial potential
- arises from ion movements
- ionic valency (z), concentration gradient (deltaCion), and ionic permeability (ease at which ion crosses membrane, Pion)
- ion movements are determined by fick’s law of diffusion
- to describe flux (J) over cell membrane, other terms are needed (Gion and Eion)
Fick’s Law of Diffusion
Movement of flux (Jion) (moles.sec-1.cm-2) = Pion - deltaCion
Problem with ficks law of diffusion
Pion is the product of the ion species and concentration gradient, and electrostatic attraction
What is Gion
the ionic conductance of the ion across the membrane (amperes) -> measure of ionic movement
What is Eion
the electrostatic diffusion potential (volts) -> measure of the size and direction of attracting forces
Transepithelial potential of potassium
- as K+ ions leave cell across chemical gradient, deltaCK diminishes
- diffusion potential (EK) increases to retain K+ in the cell
- inward/outward movement of K+ depends on the membrane permeability of K+ (PK, determined by number of pumps/channels/transporters) and is measured by movement of charge (GK)
- when EK.EK = PK.deltaCK, net flux of K+ = 0
- value of transepithelial membrane potential (Em) at which equilibrium is established is given by Nernst equation
Nernst Equation
- membrane potential at which equilibrium is established for a given ion
- Em = RT/zF ln(K[K+]o/[K+]i)
- Eion = 61log10([ion]o/[ion]i)
Value at which there is no net flux of K+ ions at 37 degrees Celcius
Em = 61.log10([5]/[155]) Volts
Em = -0.092 Volts
Em = -92mV
The Gibbs-Donnan Equilibrium
describes the effect of a non-permeable anion on transmembrane ion difference which drives water transport through osmosis
How does water move across an epithelial membrane
2 routes:
- intracellular: movement of water occurs within cells and is regulated by water channels known as aquaporins
- paracellular: movement of water occurs between cells and is regulated by tight junction permeability
What do we need for water movement across epithelial membrane
- osmotic gradient
- opening and closing of tight junctions
- aquaporins (channels specialised for the movement of water)
Mechanism of fluid secretion
- presence of a sodium and potassium pump causes sodium to drive inward fluid uptake
- chloride is moved against gradient and accumulates inside the cell (membrane potential hyperpolarises)
- adding a chloride channel (CLCN) allows chloride to exit the epithelial monolayer and enter the apical membrane
Mechanism of fluid absorption
- opening of tight junctions createsan inward current
- sodium coupled glucose uptake helps to retain fluid in cases of diarrhoea
- epithelial sodium channel (ENaC) draws fluid across apical membrane and moves it towards the blood
Role of chloride in fluid secretion
- if ENaC dominates all epithelial membranes would dry and therefore we need secretion to go in the other direction using chloride
- chloride in blood plasma has negative charge so must be coupled with strong electrochemical movement of sodium and potassium to cotransport it across the basolateral membrane
Characteristics of Cl- channels epithelial cells
fluid secretion (basolateral -> apical transport)
Characteristics of Na+ channels epithelial cells
fluid absorption (apical -> basolateral transport)
Swelling-activated Cl- channel (IClvol)
- activated transiently by osmotic shock
- sustained opening does not occur
Calcium-activated Cl- channel (CaCC)
- activated by release of intracellular Ca2+ stores
- activity is transient
- unlikely to be sustained in development
Outwardly Rectifying Cl- Channel (ORCC)
- regulated by release of intracellular ATP
- maintains cell membrane potential by regulated depolarisation to physiological set point
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
- best characterised channel due to role in CF
- long presumed to be channel regulating fluid secretion in adult lung
Voltage-dependent Cl- channels (CLCN)
- recently characterised in lung
- expression pattern follows process of lung development
The CFTR
- member of ATP binding cassette glycoprotein superfamily
- 170kDa glycoprotein Cl- channel composed of 2 6-span transmembrane domains (pore), 2 nucleotide binding domains (NBD1 and 2), and a single R domain of highly charged AAs
The R domain of the CFTR
- a regulatory site containing several phosphorylation sites for protein kinases A and C
- activation of CFTR Cl- conductance requires ATP binding to the NBD domains and phosphorylation of the R domain
Role of NBD1 and NBD2 in CFTR
- hydrolyse ATP
- provides kinetic energy to open channel
Normal cycle regulation of CFTR
1) channel is inactive
2) cAMP activation of PKA phosphorylates R domain
3) ATP binds to NBD1&2
4) ATP is hydrolysed
5) channel opens and conducts Cl-
6) dephosphorylation of the R domain inactivates the channel
Loss of Phenylalanine 508 in NBD1 region of CTFR
- causes CF
- causes CFTR to be misfolded and sticks in ER, where it is broken down and never reaches membrane
- results in no fluid in airway secretion (dry lung), sticky mucus and pathogen colonisation
- treatment for CF reinstates the appropriate folding and translocates it back to membrane
- gene therapy treatments
Voltage-dependent Cl- ion channels (CLCN 1…7)
- widely distributed (found in epithelia, muscle, nerve tissue, also plants)
- channel opening is “gated” by membrane potential
- CLCN2 expressed in the epithelium is activated at negative (hyperpolarised) cell membrane potentials
- CLCN2 and CLCN3 are developmentally expressed in the foetal lung and control lung fluid volume during development
Structure of voltage-dependent Cl- ion channels (CLCN 1…7)
- 10 transmembrane domains which dimerise to form two pores
- each pore is voltage gated
Mutations in voltage-dependent Cl- ion channels
- CLCN1 mutation: myotronia (failure of muscles to relax after contraction -> cells remain depolarised)
- CLCN5 mutation: Dent’s disease (fluid transport problems in the kidney resulting in kidney stones, calcium and protein loss in urine)
The ENaC channel
- found in all secretory epithelia
- composed of 3 subunits (alpha, beta, gamma)
- genetic knockout of alpha is lethal at birth due to flooding of lungs
- knockout of beta or gamma is not lethal but associated with reduction in rate of Na+ transport
- alphaENaC is the dominant pore-forming subunit
- association with beta and/or gamma is required to form a tetramer and confer Na+ selectivity
Structural domains of ENaC subunits
- cysteine-rich region on extracellular loop (rich in CSSC sulphur cross linking -> determines tertiary structure
- histidine glycine residue rich region involved in channel opening and closing
- proline tyrosine residue rich region which serves as a binding motif for NEDD4 (ubiquitin ligase which targets the subunit for membrane removal and proteolytic degradation)
Inhibition of ENaC
- Amiloride
- selective Na+ channel blockage shows high potency for ENaC blockers (benzamil and amiloride)
Activation of ENaC
- beta2 Adrenergic agonists (e.g. Isoproterenol)
- conductance is induced by catecholamines
Pseudohypoaldoseteronism (PHA)
- hereditary
- associated with resistance to aldosterone, leading to increased sodium excretion, dehydration, hypotension, hyperkalaemia, and metabolic acidosis
- disease is most evidence in kidney, sweat gland, pancreatic and salivary gland function
- can be lethal due to excessive hypotension and circulatory collpase
- results in excessive fluid retention in lung airways due to sustained Cl- secretion and inability to absorb Na+
- most lethal form caused by loss-of-function mutations in alpha, beta and gamma ENaC
PHA effect in renal system
- high Na/2Cl/K uptake
- high basolateral K+ secretion
- membrane removal and degradation
- Na+ secretion in urine
- ENaC channel recognized for degradation by NEDD4
Liddle’s Syndrome
- hereditary
- characterised by salt-sensitive hypertension, hypokalaemia, metabolic alkalosis, and repressed aldosterone secretion
- results from gain-of-function mutations in C-terminal domain of beta or gamma ENaC which results in deletion of 45-75 amino acids from proline-tyrosine rich PY domain
- WT PY motif binds to NEDD4 (ubiquitin ligase) promoting internalisation and degradation of subunit
- loss of repressor activity = sustained Na+ absorption
Hypernatraemic effects of Liddle’s Syndrome in Renal System
- ENac accumulation at apical membrane
- degradation of NEDD4 = sustained Na+ absorption
- increased Na+ pump activity compensates for Na+ uptake and causes Na+ secretion into blood with no route for removal
